Project Summary There is a fundamental knowledge gap in understanding the substantial variability in the incidence, onset, duration, and severity of neonatal abstinence syndrome (NAS) in infants born to women with opioid use disorder (OUD). Despite a growing body of literature in the NAS field, no single factor or combination of known factors have sufficiently explained this variability. Current clinical tools to identify infants needing pharmacological treatment for NAS are reactive rather than proactive since they focus on the severity of physical symptoms of withdrawal at onset. Thus, there is an unmet need for biomarkers in the neonate that proactively predict the severity of NAS, before the onset of physical withdrawal. The long-term goal of our program is to improve the short- and long-term outcomes of infants born to women with OUD. The objective in this particular application is to assess the extent to which intra- and extra-cellular miRNAs are sensitive to opioid withdrawal in the period preceding physiological manifestations of NAS. The central hypothesis is that umbilical cord-derived plasma and leukocyte miRNAs may be assessed as surrogate markers for brain health at birth, following opioid exposure, and predict severity of NAS before the emergence of physiological signs of withdrawal. Our preliminary data indicate that important brain miRNAs are secreted by developing neural cells and present in neonatal plasma, and can serve as a biomarker for brain effects of prenatal drug exposure. Guided by these preliminary data, the hypothesis will be tested by pursuing the specific aim to identify an infant miRNA signature that precedes and predicts NAS. This will be assessed in 70 well-characterized infants prenatally exposed to opioids by identifying 1A) intra-cellular (leukocytes) and extra-cellular (plasma) miRNAs that differentiate infants with NAS (cases) and without NAS (controls); 1B) the extent to which this miRNA composite signature of cell and tissue adaptation predict the severity of NAS. The rationale for the proposed research is that miRNAs can be used not only as diagnostic biomarkers for drug exposure and effects, but also can be manipulated to diminish effects of drug exposure and to provide neuroprotection. The approach is innovative, because it examines a unique set of biomarkers within the context of other perinatal predictors of infant health and will leverage resources of an established, well-characterized longitudinal cohort. The proposed research is significant because it is expected to lead to better understanding of maternal, newborn, and epigenetic factors contributing to NAS variability, and is expected to lead to identification of new therapeutic targets for preventing NAS. Better understanding of mechanisms involved in NAS pathophysiology has the potential to change clinical management of NAS, which now affects 6 per 1000 hospital births. Ultimately, such knowledge has the potential to improve short- and long-term outcomes of the growing number of infants prenatally exposed to opioids in the U.S.